The morphological characteristics of strain NEAU-YY56T showed that the strain had the typical characteristics of the genus Cellulomonas. The cells of NEAU-YY56T were found to be Gram-positive, motile and rod-shaped about 1.6-1.7 μm in length and 0.3-0.4 μm in width (Fig. 1). Colonies are smooth, circular and pale yellow on TSA medium. Strain NEAU-YY56T was observed to grow moderately on TSA, ISP 2 and ISP 3 agar media and poorly on ISP 4, ISP 5, ISP 6 and ISP 7 agar media. No diffusible or melanoid pigments were observed on the tested media. The colonies were in yellow-color-series, cultural characteristics of the isolate are shown in Table S1. The strain was found to grow at a temperature range of 18-40 oC (optimum temperature 28 oC), pH 6.0-8.0 (optimum pH 7.0) and tolerated 0-3.0 % NaCl (optimum NaCl of 0 %). A decolorizing circle was observed around the colony of strain NEAU-YY56T on azure B plate, indicating that the strain had lignin degradation ability with a degradation diameter of 29.2 mm (Fig. S1). Certain data of physiological and biochemical characteristics could clearly distinguish strain NEAU-YY56T from its closely related strains, C. pakistanensis JCM 18755T, C. denverensis W6929T and C. hominis JCM 12133T (Table 1). For instance, strain NEAU-YY56T could not grow at pH 9.0, while the closely related strains could. The tolerance to NaCl of strain NEAU-YY56T was up to 3.0 %, which was lower than the closely related strains. Other phenotypic differences include the decomposition of aesculin, hydrolysis of starch, reduction of nitrate and patterns of carbon and nitrogen utilization. Detailed physiological and biochemical characteristics are presented in the species description.
Strain NEAU-YY56T was found to exhibit a range of chemotaxonomic properties that are consistent with the description of the genus Cellulomonas. Whole-cell sugars contained glucose, rhamnose and ribose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside and two unknown glycolipids (Fig. S2). The predominant menaquinones were MK-9(H4) (56.1 %) and MK-9(H2) (43.9 %). The cellular fatty acids of strain NEAU-YY56T were anteiso-C15:0 (37.1 %), C16:0 (28.3 %), C14:0 (15.4 %), anteiso-C17:0 (9.4 %), iso-C16:0 (4.0 %), C18:0 (3.2 %), C17:0 (1.2 %), C18:1ω9c (0.9 %), C16:1ω9c (0.4 %) and C19:0 (0.1 %). The DNA G+C content of strain NEAU-YY56T was 74.7 %. All these chemotaxonomic data showed that strain NEAU-YY56T should be assigned to the genus Cellulomonas.
Identification using the EzBioCloud server revealed that strain NEAU-YY56T belongs to the genus Cellulomonas and shared high sequence similarities with C. pakistanensis JCM 18755T (98.7 %), C. denverensis W6929T (98.2 %) and C. hominis JCM 12133T (98.1 %). In addition, the phylogenetic trees based on 16S rRNA gene sequences (1,446 bp) reconstructed using the neighbour-joining (Fig. 2) and maximum-likelihood (Fig. S3) algorithms showed that strain NEAU-YY56T formed a cluster with C. pakistanensis JCM 18755T, C. denverensis W6929T and C. hominis JCM 12133T, which was further confirmed by the phylogenetic tree based on the concatenated sequences (3,594 bp) of the partial sequences of housekeeping genes including gyrB, rpoB, recA, relA and atpD reconstructed by the neighbour-joining (Fig. 3) and maximum-likelihood (Fig. S4) algorithms. Thus, based on these phylogenetic relationships and 16S rRNA sequences similarities, the isolate was grouped with members of the genus Cellulomonas.
The genome sequence of the assembled strain NEAU-YY56T was 4,391,169 bp and has a DNA G+C content of 74.7 %. Also, it was made up of 280 scaffolds with an N50 of 30,351 bp. The genome sequence was deposited in the GenBank/EMBL/DDBJ with the accession number RFFI00000000. And NCBI Prokaryotic Genome Annotation Pipeline (PGAP) showed that there were three copies of 5S rRNA genes, four copies of 16S rRNA genes, one copy of 23S rRNA gene, 48 tRNA genes, three copies of noncoding RNA genes. More specific genomic information and obvious features are presented in Table 2. AntiSMASH 6.0 was used genome mining analysis led to the identification of 9 putative gene clusters responsible for the production of diverse secondary metabolites. Of which 8 putative gene clusters displayed low similarity to the known gene clusters of tylactone, terpene and so on. And one putative gene cluster was 100% similar to alkylresorcinol. Of the genome of 4,174 genes, 3,294 genes were annotated and classified into known functions according to the KEGG database. By analyzing the genome of the strain NEAU-YY56T, there were 855 genes involved in metabolism-associated pathways. Genome analysis of the COG database revealed that strain NEAU-YY56T contained 272 amino acid transport and metabolism genes, 413 carbohydrate transport and metabolism genes, 335 transcription genes, 222 inorganic ion transport and metabolism genes and 299 general function prediction only genes. Moreover, the genomic analysis of the COG database showed that strain NEAU-YY56T contained a gene encoding laccase (GM002387) and a gene encoding catalase (GM003718), both of which are key enzymes in lignin degradation (Blánquez et al. 2017; Zhang 2006). Therefore, the genomic analysis also confirmed that strain NEAU-YY56T could decompose lignin.
ANI value and dDDH level were employed to determine whether the strain could be considered to represent a new species. The ANI values between strain NEAU-YY56T and its reference strains, C. pakistanensis JCM 18755T, C. denverensis W6929T and C. hominis JCM 12133T, were 83.5 %, 79.0 % and 83.1 %, respectively, which were lower than the 95 %-96 % threshold defined by prokaryotic species (Richter and Rossello-Mora 2009; Chun and Rainey 2014). Similarly, the low dDDH levels between strain NEAU-YY56T and C. pakistanensis JCM 18755T (26.8 ± 2.4 %), C. denverensis W6929T (21.8 ± 2.3 %) and C. hominis JCM 12133T (26.2 ± 2.4 %) were also well below the threshold (70 %) recommended by Wayne et al. (1987) for assigning strains to the same genomic species. These results supported the conclusion that strain NEAU-YY56T represents a novel species of the genus Cellulomonas.
Therefore, based on the distinct morphological, physiological and biochemical, chemotaxonomic and phylogenetic and DNA-DNA relatedness data mentioned above, strain NEAU-YY56T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas triticagri sp. nov. is proposed.
Description of Cellulomonas triticagri sp. nov.
Cellulomonas triticagri (tri.tic.a’gri. L. neut. n. triticum wheat; L. masc. n. ager field; N.L. gen. n. triticagri of a wheat field)
Short rods, about 1.6-1.7 μm in length and 0.3-0.4 μm in width. Gram-stain positive, aerobic and motile actinomycete that produces pale yellow and smooth mycelium after 2 weeks at 28 °C on TSA medium. Moderate growth is observed on TSA, ISP 2 and ISP 3 agar media; poor growth on ISP 4, ISP 5, ISP 6 and ISP 7 agar media. Colonies are in yellow-color-series. No diffusible or melanoid pigments are observed on the tested media. Grows at 18-40 ℃ (optimum 28 ℃), at pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v, optimum 0 %) NaCl. Hydrolysis starch and Tweens (20, 40 and 80), but not aesculin. Catalase is produced, but H2S and urease are not. Besides that, negative for decomposition of cellulose, peptonization and coagulation of milk, reduction of nitrate and liquefaction of gelatin. D-Fructose, meso-inositol, lactose, D-maltose, D-mannose, L-rhamnose and D-sorbitol are utilized as sole carbon sources, but not L-arabinose, dulcitol, D-galactose, D-glucose, D-mannitol, D-raffinose, D-ribose, D-sucrose or D-xylose. L-Arginine, L-glutamic acid, L-glutamine and L-tyrosine are utilized as sole nitrogen sources, but not L-alanine, L-asparagine, L-aspartic acid, creatine, glycine, L-proline, L-serine or L-threonine. Whole-cell sugars contain glucose, rhamnose and ribose. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside and two unknown glycolipids. The predominant menaquinones are MK-9(H4) and MK-9(H2). The predominant cellular fatty acids (> 5.0 %) are anteiso-C15:0, C16:0, C14:0 and anteiso-C17:0. The G+C content of the DNA of the type strain is 74.7 %.
The type strain is NEAU-YY56T (= DSM 106717T = JCM 32550T), was isolated from the rhizosphere soil of wheat (Triticum aestivum L.) collected from Zhumadian, Henan Province, Central China. The GenBank accession numbers for the 16S rRNA gene sequence and the draft genome sequence of the type strain are MH144588 and RFFI00000000, respectively.